Urinary tract infections (UTI) are prevalent bacterial infections that originate in the bladder and can ascend to the kidney, leading to acute kidney injury and long term sequelae such as renal scarring and chronic kidney disease. To protect the kidney from acute infection and its sequelae, our long term goal is to devise strategies to prevent UTI by promoting host innate immunity. Emerging data point to a key role for host antimicrobial peptides (AMPs) in shielding the urinary tract from invading microbes. Our research group has identified RNase 6 as a monocyte and macrophage-derived AMP with inducible expression during UTI in humans and mice. RNase 6 exhibits potent antimicrobial activity toward uropathogenic bacteria and absent cytotoxicity toward host cells. However, the specific roles and regulation of RNase 6 during experimental UTI remain unknown. This knowledge gap limits the development of RNase 6 as a novel therapeutic agent to combat UTI. To investigate the regulation and contributions of RNase 6 during experimental UTI, we have engineered mice with a Rnase6EGFP knock-in allele. In Specific Aim 1, we propose experiments to determine the consequences of Rnase6 deletion on host UTI susceptibility and to clarify the cellular sources of RNase 6 during UTI. In Specific Aim 2, we will determine the specific contributions of RNase 6 to macrophage antimicrobial activity, and clarify the mechanisms responsible for macrophage RNase 6 production in response to uropathogenic bacteria. Completion of these studies will advance our knowledge of RNase 6 function and regulation during UTI, and give impetus to the development of AMP-based therapeutic strategies to prevent UTI.